Method of obtaining antibodies

ABSTRACT

Antibodies specific to glycosylated hemoglobin and substantially free of cross-reactivity with nonglycosylated forms of hemoglobin are obtained by administering a synthetic antigen to a host animal. The antigen comprises a glycosylated synthetic peptide having an amino acid sequence corresponding to that of the NH 2  -terminus of the β-chain of hemoglobin. The peptide comprises a sequence of between about four and ten, and preferably seven, amino acids, and is conjugated with glucose and a protein. The protein preferably comprises an immunoglobulin which is foreign to the host animal.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of obtaining antibodies and, morespecifically, this invention relates to a method of obtaining anantibody which is highly specific to glycosylated hemoglobin andsubstantially free of cross-reactivity with non-glycosylated forms ofhemoglobin.

2. Description of the Prior Art

Many antigenic molecules which are clinically important are not subjectto determination by immunoassay due to their molecular structure.Specifically, some antigens comprise large molecules having manyantigenic sites and, as a result, antibodies produced from such antigensmay exhibit a high degree of cross-reactivity with respect to variousspecies of antigens, and thus are of only limited utility in immunoassaytechniques.

Hemoglobin Alc (HbAlc) has an amino acid structure which is identical tothat of the HbAo form of hemoglobin, except for the presence of aglucose moiety attached to the NH₂ -terminus valine in the β-chain. Thetransformation of HbAo to HbAlc is a continuous process which is afunction of blood glucose concentration. The level of HbAlc in a bloodsample is an indication of an individual's carbohydrate metabolism.

Normal adults have about 90% of their total hemoglobin as HbAo, 2-3% asHbAla and HbAlb, and 3-6% of their total hemoglobin as HbAlc. However,the level of HbAlc in juvenile and maturity onset diabetics ranges fromabout 6% to about 15%. The level of HbAlc in hypoglycemic patients iscorrespondingly less than about 3%.

The quantification of the HbAlc level in diabetic patients is a usefulmeans of assessing carbohydrate tolerance as well as adequacy ofcontrol. One prior method of determining the HbAlc level in a serumsample is the well-known column chromotography method wherein the sampleis eluted through a column. Glycosylated hemoglobin is eluted prior tononglycosylated hemoglobin.

However, the results of prior art column chromotography methods aresensitive to many variables, specifically including temperature, columnlength and ionic strength effects encountered in the column. Priorcolumn methods require pre-separation steps, are not homogeneous, andare relatively nonspecific because they measure HbAo, HbAla, HbAlb, andHbF in addition to HbAlc.

Prior methods of producing antibodies to glycosylated hemoglobin for usein immunoassay procedures involve separation of glycosylated hemoglobinfrom serum, and production of antibodies by injection of theglycosylated hemoglobin fraction into a host animal. Resultingantibodies are then harvested and labeled, as with a radioactive label,and utilized in suitable assay methodologies, such as RIA.

Exemplary of such prior methods is the work of Cerami and co-workersdescribed in U.S. Pat. No. 4,247,533 (Jan. 27, 1981) and in Javid et al,"Immunologic Characterization and Qualification of Haemoglobin A_(lc) ",British Journal of Haemotology, Vol. 38 at 329-337 (1978).

Such prior assay methods have generally been unsuccessful, since thehemoglobin molecule is very large, having many antigenic sites, withonly minor structural differences between glycosylated andnonglycosylated hemoglobin. Hence, prior assay methodologies havegenerally been characterized by low specificity due to cross-reactivityof antibodies produced by injection of HbAlc with various forms ofnonglycosylated hemoglobin. See Chou et al, "Development of a LaserNephelometric Method for the Quantitation of Human Glycohemoglobins",Analytical Letters, Vol. 14 (B13) at 1071-1087 (1981).

SUMMARY OF THE INVENTION

It is an object of the invention to overcome one or more of the problemsdescribed above.

According to the invention, antibodies which are highly specific to aselected antigen are obtained by immunization of a host animal with asynthetic analog of the antigen coupled to a carrier protein.

The antigenic analog is prepared by synthesizing an amino acid sequencecorresponding to that of a portion of the antigen and of sufficientlength to elicit an authentic immune response when administered to thehost, yet insufficient to elicit production of antibodies which exhibitcross-reactivity to species other than the selected antigen.

Prior to immunization of the host, the synthetic amino acid sequence iscoupled to a carrier protein, preferably through a covalent linkage.

More specifically, antibodies against glycosylated hemoglobin(specifically HbAlc) which are as substantially free of cross-reactivityto nonglycosylated hemoglobins (specifically, hemoglobins other thanHbAlc) are obtained by synthesis of a suitable peptide, glycosylationand coupling of the peptide to a carrier protein, administration of theresulting synthetic antigen to a host animal, and harvesting of theresulting antiserum from the animal.

In a preferred embodiment, the antigen is prepared by first synthesizinga hapten consiting of a peptide having an amino acid sequencecorresponding to the NH₂ -terminus of the hemoglobin β-chain. The haptencomprises a sequence of between about four and ten, and preferablyseven, of the amino acids which comprise the NH₂ -terminus of thehemoglobin β-chain.

After synthesis, the peptide is glycosylated, preferably with ad-glucose, and conjugated, preferably by covalent linkage, with asuitable carrier protein.

After preparation, the glycosylated peptide-protein antigen isadministered to a suitable host animal, and the resulting antiserum isharvested.

The protein is preferably an immunoglobulin which is foreign to the hostanimal, and the animal is preferably one whose metabolism does notnaturally produce HbAlc. For example, the protein may be bovine IgG, andthe host animal may be a sheep.

The resulting antibodies to the synthetic antigens are highly specificagainst HbAlc and thus may be used in any of various forms ofimmunoassay, including RIA, ELISA or EMIT immunoassay methodology, forexample.

Thus, the invention also provides a method of quantitatively detectingthe presence of HbAlc in a sample. This method is especially suitablefor use in assessing carbohydrate intolerance in a living mammal.

A diagnostic test kit embodying the inventive antibodies, in combinationwith a suitable labeled antigen control, is also described.

Other objects and advantages of the invention will be apparent from thefollowing detailed description of the preferred embodiments, taken inconjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elution profile of the iodinated antigen (HbAlc) fractionsproduced in Example 6; and,

FIG. 2 is a curve of an antibody titer of antiserum produced accordingto the invention.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention will be illustrated by reference to aspecific application of the method to production of antibodies which arehighly specific to HbAlc. It is to be understood, however, that theinvention is not limited by reference to a specific application but isintended to apply to production of antibodies which are respectivelyhighly specific to a wide variety of antigens.

Glycosylation of hemoglobin in both humans and animals occurs at the NH₂-terminus of the human hemoglobin β-chain of the hemoglobin molecule.The NH₂ -terminus of the human hemoglobin β-chain is characterized by aspecific amino acid sequence, as follows:

    NH.sub.2 --Val--His--Leu--Thr--Pro--Glu--Glu--Lys--Ser--Ala--

The invention contemplates the chemical synthesis of a peptideconsisting of a sequence of amino acids corresponding to that of all ora portion of the foregoing hemoglobin β-chain NH₂ -terminal end. Thepeptide comprises between about four and ten, and preferably seven, ofthe foregoing amino acids in the sequence given.

After synthesis, the peptide is glycosylated at the terminal valine,which corresponds to the NH₂ -terminus of the hemoglobin β-chain and isconjugated, preferably by means of a covalent linkage, with a suitablecarrier protein, such as an immunoglobulin. The peptide may beglycosylated prior or subsequent to conjugation.

When administered to a host animal, the glycosylated and conjugatedpeptide promotes the formation of antibodies which, when harvested, maybe utilized in immunoassay techniques for the quantitative andqualitative determination of glycosylated hemoglobin in a sample. Theantibodies to the synthetic antigen are highly specific against HbAlcand are substantially free of cross-reactivity against other forms ofhemoglobin.

The highly-specific hemoglobin Alc antibodies may be incorporated in adiagnostic test kit in either lyophilized or solution form, as describedbelow.

Synthesis of Peptide

One disadvantage of utilizing pure hemoglobin Alc as an antigen topromote antibody production is that the hemoglobin Alc molecule is quitelarge and comprises many antigenic sites. In connection with thisinvention, a synthetic peptide having an amino acid sequencecorresponding to that of the NH₂ -terminus of the hemoglobin Alc β-chainis utilized, thus minimizing or substantially eliminating the productionof antibodies which are nonspecific to hemoglobin Alc.

The synthesis of peptides comprising various selected sequences of aminoacids is well known. One peptide synthesis method especially suitablefor use in connection with this invention is described in Merrifield, R.B., "Solid Phase Peptide Synthesis.I.The Synthesis of a Tetrapeptide",JACS Vol. 85 p. 2149 (1963), the disclosure of which is herebyincorporated by reference. Numerous modifications of the Merrifieldmethod have been published and are suitable for use in connection withthis invention.

In this connection, an amino sequence of suitable length is prepared,preferably according to the procedure of Merrifield, above. Peptidescomprising between about four and ten amino acids in the sequence of thehemoglobin β-chain NH₂ -terminus are suitable for use in promotingantibody production. A peptide having a sequence of seven amino acids ispreferred.

Thus, the synthetic peptide used in this invention has the followingamino acid sequence:

    Val--His--Leu--Y

where Y is chosen from the group consisting of Thr--, Thr--Pro--,Thr--Pro--Glu--, Thr--Pro--Glu--Glu--, Thr--Pro--Glu--Glu--Lys--,Thr--Pro--Glu--Glu--Lys--Ser--, and Thr--Pro--Glu--Glu--Lys--Ser--Ala--.Preferably, the peptide is a sequence of seven amino acids, and the Ycomponent comprises Thr--Pro--Glu--Glu--.

A suitable synthetic peptide having a desired amino acid sequence, andprepared according to the Merrifield procedure can be obtained fromPeninsula Laboratories, P.O. Box 1111, San Carlos, Calif., 94070 (USA).

Example 1, below, illustrates the Merrifield procedure for preparing thepreferred peptide of this invention.

Glycosylation of Peptide

The synthetic peptide prepared according to the foregoing isglycosylated by addition of glucose, preferably d-glucose, to theterminal valine thereof.

Several methods for carrying out such glycosylation are known. One suchmethod is described by Brownlee et al, "A Glucose-Controlled InsulinDelivery System: Semisynthetic Insulin Bound to Lectin", Science, Vol.206, pp. 1190-1191 (1979), the disclosure of which is herebyincorporated by reference. The Brownlee et al glycosylation methodcomprises simple incubation of d-glucose and a peptide in a 0.1 M NaPO₄buffer, pH=8.0, at 37° C. for a reaction period of up to about ten days.

A preferred method of glycosylating a synthetic peptide is described inDixon, "A Reaction of Glucose with Peptides", Biochem. J., Vol. 129, pp.203-208 (1972), the disclosure of which is hereby incorporated byreference.

According to the Dixon method a 1:1 (v/v) mixture of pyridine andglacial acetic acid is added to a mixture of peptide and d-glucose in asuitable container. The reaction mixture is stirred at ambienttemperature for a suitable period of time, typically several days.

After reacting, the mixture is placed in a freeze-dryer and solvent isevaporated to dryness. The resulting almost dry material may be refinedas set forth in Example 2, below.

The glycosylated peptide may be characterized by silica gel thin layerchromatography, or by other suitable characterization methods, in orderto assure that the desired glycosylated peptide is obtained.

Glycosylation of peptide according to the Dixon method has been found toresult in at least about 95% glycosylation of the peptide. The Brownleeet al method, on the other hand, while useful, typically results only inabout 50% glycosylation.

Conjugation of Glycosylated Peptide with Protein

In accordance with the invention, and before or after glycosylation, thepeptide prepared as described above is conjugated at its Y-terminus witha carrier protein such that, when the conjugated peptide is administeredto a host animal, the conjugated peptide acts as an antigen to promotethe production of antibodies which are subject to harvesting.

Any of a wide variety of proteins may be used as a carrier for thepeptide. It is preferred, however, that the protein be one which isforeign to the host animal.

For example, if the host animal is a sheep, a suitable protein is onewhich as been derived from a cow, or another suitable animal other thana sheep.

The protein carrier is preferably a relatively large molecule, such asbovine albumin, rabbit albumin, alkaline phosphatase, or others, inorder to enhance the antigenic capability of the conjugated peptide. Apreferred type of protein carrier is immunoglobulin-G, preferably bovineIgG when the host animal is a sheep.

The glycosylated peptide is linked to the carrier at the peptideY-terminus by any of a variety of methods. The peptide is preferablycovalently linked with the protein.

One preferred method of conjugating a peptide with a protein isdescribed in Goodfriend et al., "Antibodies to Bradykinin andAngiotensin: A Use of Carbodiimides in Immunology", Science, Vol. 144,pp. 1344-1346 (1964), the disclosure of which is hereby incorporated byreference.

While the Goodfriend et al article describes a method of covalentlylinking a peptide to a protein with a carbodiimide linkage, it is to beunderstood that other types of linkages may be used. Suitable linkingagents including glutaraldehydes, N-N-carbonyldiimidazole,1-hydroxybenzotriazole monohydrate, N-hydroxy succinimide,n-trifluoroacetylimidazole, or cyanagen bromide.

Briefly stated, the Goodfriend et al method involves a reaction betweena protein such as bovine IgG, a peptide and a carbodiimide, such as1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride ("EthylCDI") in water at ambient temperature. A reaction production in the formof a precipitate and a colloidal suspension is obtained. The reactionmixture is dialyzed against distilled water at low temperatures and,after dialysis, the reaction mixture may be lyophilized, or used insolution form.

Production of Antibodies

The antigen obtained as described is administered to a host animal, suchas a goat, sheep, etc. by any suitable method and resulting antibodiesare harvested. Usually, an adjuvant, preferably Freund's complete orincomplete adjuvant, is administered with the antigen to promoteantibody production.

The host animal is preferably one whose metabolism does not naturallyproduce hemoglobin Alc.

Immunoassay Utilizing HbAlc-Specific Antibodies

The antibodies obtained as described above are highly specific to HbAlc,and are useful in any of a wide variety of immunoassay techniques,including RIA, enzyme immunoassay techniques such as ELISA and EMITsystems, and fluorescent immunoassay techniques.

The HbAlc-specific antibodies are conveniently incorporated into adiagnostic test kit wherein the antibodies are packaged in a container,either in lyophilized or solution form, with a separate containercontaining radioactive or enzyme-labeled hemoglobin Alc antigen controlmaterial. The control material may comprise antigen produced accordingto the invention.

The following specific examples are given for purposes of illustration,and are to be considered preferred forms of the invention. However, nounnecessary limitations are to be inferred from the examples, as obviousvariations in procedure and constituents will be obvious to thoseskilled in the art.

Example 1--Preparation of Synthetic Peptide

A synthetic peptide having the following sequence:

    Val--His--Leu--Thr--Pro--Glu--Glu--

is prepared according to the method of Merrifield, above, using anautomatic Schwarz/Mann synthesizer, as follows:

Boc--Glu(OBzl)-resin (1 mM/2 g of resin) is used as the solid support.The following Boc-protected amino acids are successively added to theBoc--Glu--(OBzl)-resin: Glu(OBzl), Pro, Thr(Bzl), Leu, His(Tos) and Val.A 3.0 molar excess of each protected amino acid is used. The success ofthe coupling reaction is monitored by the semi-quantitative ninhydrintest.

The following steps are used to couple each Boc-amino acid to theBoc--Glu(OBzl)-resin:

1. washing with CH₂ Cl₂ (1×50 ml)

2. prewashing with 35% CF₃ COOH in CH₂ Cl₂ (1×50 ml)

3. deprotection with 35% CF₃ COOH in CH₂ Cl₂ (1×50 ml, 20 min)

4. washing with CH₂ Cl₂ (2×50 ml)

5. washing with EtOH (1×50 ml)

6. washing with CH₂ Cl₂ (2×50 ml)

7. prewashing with 10% Et₃ N in CH₂ Cl₂ (1×50 ml)

8. neutralization with 10% Et₃ N in CH₂ Cl₂ (1×50 ml), 10 min.)

9. washing with CH₂ Cl₂ (3×50 ml)

10. protected amino acid (3.0 molar excess) in CH₂ Cl₂ (50 ml) is added

11. DCC in CH₂ Cl₂ (0.5 M, 6 ml) is added (the reaction time is up to 2hr.)

12. washing with CH₂ Cl₂ (3×50 ml)

The resulting protected heptapeptide,Boc--Val--His(Tos)--Leu--Thr(Bzl)--Pro--Glu(OBzl)-resin is washed wellwith 35% CF₃ COOH in CH₂ Cl₂, CH₂ Cl₂ and MeOH respectively. Afterdrying in vacuo overnight, the peptide resin is cleaved by HF (30 ml) inthe presence of anisole (10 ml) for one hour at 0° C. The reactionmixture is dried in vacuo and washed with anhydrous ether. The desiredheptapeptide is dissolved in 10% HOAc and the resin is filtered off. Thefiltrate is lyophilized to give crude Val--His--Leu--Thr--Pro--Glu--Glu.

This peptide is purified by counter-current distribution usingn-BuOH:HOAc:H₂ O (4:1:5 v/v) as the partition solvent, affording pureVal--His--Leu--Thr--Pro--Glu--Glu (450 mg).

Characterization of the peptide is carried out as follows:

Thin-layer chromatography: R_(f) 0.28 (silica gel plate;n-BuOH:HOAc:EtOAc:H₂ O ratio of 1:1:1:1; ninhydrin spray) and R_(f) 0.24(silica gel plate; n--BuOH:Pyridine:HOAc:H₂ O ratio of 15:10:3:12;ninhydrin spray).

Electrophoresis: Whatman 3 MM paper, pH 5.6 pyridine-acetate buffer,1000 V, one hour. R_(f) 0.16 with reference to picric acid.

Amino acid anlaysis: 6 N HCl at 110° C. for 72 hours. His 0.98, Thr0.92, Glu 2.10, Pro 1.00, Leu 1.03 and Val 0.97.

Example 2--Glycosylation of Peptide

The peptide of Example 1 is glycosylated with d-glucose according to themethod of Dixon et al., above, as follows:

16.4288 mg of the peptide of Example 1, in the form of a lyophilized,fluffy white light powder (MW 821.44) and 5.50 mg of anhydrous d-glucose(C₆ H₁₂ O₆ Mallinkrodt, Inc. Product No. 4912, Lot No. WAPR, MW 180.16)are introduced to a 4 ml glass vial. 1 ml of a 1:1 (v/v) mixture ofpyridine and glacial acetic acid is added to the glass vial to slowlydissolve the contents. The contents are then stirred magnetically atroom temperature for 144 hours.

The reaction mixture is then placed in a freeze-dryer for about 26 hoursand dried in vacuo to result in a brown colored, sticky material whichis nearly dry. This material was dissolved in 1.5 ml methanol and thenprecipitated slowly by addition of anhydrous diethyl ether. The mixturewas maintained at 4° C. overnight.

The precipitate was then collected by centrifugation (400 rpm, 4° C.) inglass tubes. The supernatant liquids were pipetted off and theprecipitates were initially dried under a thin stream of nitrogen, thenfull dried under vacuum for 4 hours. The dried material was dissolved in2 ml distilled water and then lyophilized.

The lyophilized material was characterized by paper electophoresis usingPauly reagent as a glycosylated peptide having the following structure:

    d-glucose--Val--His--Leu--Thr--Pro--Glu--Glu--

It was found that over 95% of the peptide introduced to the reaction wasglycosylated. A minor product was a double glycosylated form of thepeptide having the following probable structure:

    d-glucose-d-glucose--Val--His--Leu--Thr--Pro--Glu--Glu--

Example 3--Conjugation of Glycosylated Peptide with Bovine GammaGlobulin

The glycosylated peptide of Example 2 is conjugated with bovine IgGaccording to the procedure of Goodfriend et al, above, as follows:

Approximately 14 mg of bovine gamma globulin (Sigma No. G-3500, Lot No.89C-0063-1) and 28 mg of the glycosylated peptide of Example 2 weredissolved together in 0.75 ml of distilled water.

To this mixture was added 0.4 ml of distilled water containing 250 mg offreshly dissolved 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride ("Ethyl CDI") (Sigma No. E-7750, Lot No. 69C-0297). Thereaction was permitted to proceed with gentle rotation for 60 minutes.The pH as checked with pH paper was between 6 and 8.

Formation of a precipitate and a colloidal suspension was noted. Theentire reaction mixture was dialyzed for 24 hours against distilledwater at 4° C. with five changes of water (1 liter of distilled waterwas added at each change).

After 24 hours of dialysis, the entire reaction mixture was transferredto glass vials and lyophilized over a 3-day period. The resultingproduct is a dry, light and fluffy brown powder weighing 17 mg.

The conjugation reaction is preferably carried out at a pH of betweenabout 3 and 8, but a pH outside of the range is acceptable, although theyield may be lower. A pH of about 7 is preferred.

The reaction may be carried out at a wide range of temperatures (e.g. 2°C.-56° C.), but room temperature or below is preferred. 4° C. isespecially preferred.

The mole ratio of peptide to carrier protein is widely variable, anddepends primarily on the number of binding sites present on the proteinmolecule.

Example 4--Immunization of Host Animal

Two sheep were obtained through Tago, Inc. of Burlingame, Calif.Non-immuned serum is obtained from each sheep before immunization.

5.0 mg of the glycosylated, conjugated peptide (antigen) of Example 3 isdissolved in 6 mL of saline(0.9%) solution, and 6 mL of Freund'sComplete Adjuvant is added to form an emulsion. Each sheep is immunizedby intradermal injection at 12 sites using a total of 6 mL emulsion (2.5mg antigen) per sheep.

Booster injections are carried out 21 days, 34 days, and 62 daysfollowing the initial inoculations. For the first booster injection, anemulsion comprising 4.0 mg of the antigen of Example 3, 6.0 mL of 0.9%saline solution, and 6.0 mL of Freund's Incomplete Adjuvant is used.Each sheep is injected intradermally with a total of 6.0 mL of theemulsion at at least 10 sites.

For the second booster injection, an emulsion comprising 4.0 mg of theantigen, 5.0 mL of 0.9% saline solution, and 5.0 mL of Freund'sIncomplete Adjuvant is used. Each sheep is injected intradermally with atotal of 4.0 mL of the emulsion at at least 10 sites.

The emulsion used in the third booster injection comprises 3.0 mg of theantigen, 5.0 mL of 0.9% saline solution and 5.0 mL of Freund'sIncomplete Adjuvant. Each sheep is injected intradermally with a totalof 5.0 mL of the emulsion at at least 10 sites.

Example 5--Detection of Antibodies to HbAlc

A test bleeding of the sheep of Example 4 is carried out 61 days afterthe initial immunization (1 day prior to the third booster injection),and a sample of blood subjected to the well-known Ouchterlony DoubleDiffusion Technique to detect the presence of antiserum to HbAlc. (Thetesting was carried out at room temperature for 72 hours, using pureHbAlc (lmg/mL in 0.9% saline) and pure HbAo (1 mg/mL in 0.9% saline).The diffusion plates are then rinsed with 0.9% saline (with threechanges) for 72 hours, and rinsed with distilled water (with threechanges) for one day. The plates are then dried at 56° C. overnight andstained.)

The results clearly indicate the presence of antibodies to HbAlc.

The antibodies obtained as described above are identified as beinghighly specific to HbAlc, and are useful in effecting immunoassay ofHbAlc in any of a variety of body fluids according to any convenientimmunoassay technique.

The antiserum obtained as described above is highly specific to HbAlc,as demonstrated by the following Examples 6 and 7.

Example 6--Radioactive Labeling of Antigen

Radioactive labeled indexed antigen (HbAlc) is prepared by thewell-known Chloramine T procedure, as follows:

Sephadex is swelled in 0.01 M PBS at room temperature overnight, and acolumn is packed in a 10 ml disposible pipette (gel bed volume=about 10ml). The column and bed are then washed with 50 ml PBS, and the columnis equilibrated with 20 ml of an elution buffer.

(The elution buffer solution comprises 0.01 M sodium phosphate buffer(pH 7.5), 0.15 M NaCl, 0.25% (w/v) bovine serum albumin, 0.01% (w/v)EDTA (disodium salt), and 0.01% (w/v) NaN₃.)

The concentration of substantially pure HbAlc in 0.01 M sodium phosphatebuffer (pH 7.5) is adjusted to 1 μg/μL, and 5 μL (5 μg) of the resultingsolution is introduced to a Pierce Reacti-Vial® reaction vial. 25 μL of0.5 M sodium phosphate buffer (pH 7.5) is added, along with 1 mciNaI-125. 5 μL chloramine T is added, and the vial is capped and gentlymixed.

The reaction is allowed to proceed for about 15 seconds, and 25 μLsodium metabisulfite solution (6.3 mg in 10 mL 0.05 M sodium phosphatebuffer (pH 7.5)) is added to stop the reaction.

The reaction mixture is applied to the Sephadex column, and the vial isrinsed with 50 μL of elution buffer solution. The elution buffer is thencarefully applied to the column, and the flow rate adjusted to 10seconds/drop.

A total of 40 fractions (10 drops/fraction) are collected and 1 μL fromeach fraction is taken for readioactive counting to obtain an elutionprofile, shown in FIG. 1.

As can be clearly seen in FIG. 1, iodinated antigen fractions areseparate from fractions containing free iodine.

The fractions containing iodinated HbAlc are then pooled, and aresuitable for dilution or concentration, depending upon the intended useof the labeled HbAlc.

(The quality of the labeled HbAlc can be quickly checked byprecipitating aliquots thereof with trichloroacetic acid (5%), andcounting the radioactivity of the precipitate and supernatant liquid,respectively. Normally, over 90% of the radioactivity will beprecipitatd; otherwise, the quality of the labeled HbAlc should beregarded as poor).

Example 7--Assay of Antibody Titer

The ¹²⁵ I-labeled HbAlc of Example 6 is diluted in a dilution buffersolution which comprises 0.01 M sodium phosphate buffer (pH 7.5), 0.15 MNaCl, 1 mM EDTA (disodium salt), 0.1% (w/v) NaN₃, and 2% (w/v) bovineserum albumin. The activity of the diluted labeled antigen is about20-30 KCPM/200 μL.

Rabbit anti(sheep IgG) is diluted in the dilution buffer at 1:200.Polyethylene glycol is added to a total of 6%.

Antiserum obtained in Examples 4 and 5 is diluted (in series) in normalsheep serum (serum obtained from the sheep of Example 4 prior to theinitial immunization).

200 μL of dilute HbAlc solution is placed in each of a series of testtubes, and 100 μL of each antiserum dilution, each containing betweenzero and 31 μL, is added to the tubes. (e.g. 100 μL of a dilutioncontaining 20 μL of antiserum contains 80 μL normal sheep serum.)

The tubes are incubated at room temperature for 12-16 hours, and 0.7 mLof the rabbit anti(sheep IgG) dilution is added to each tube. The tubesare then vortex-mixed, incubated at room temperature for 15 minutes,centrifuged and the supernatant liquids decanted.

The radioactivity of the precipitate in each tube is counted in a γcounter, using normal sheep serum as the blank. Results are shown inFIG. 2.

FIG. 2 clearly shows that an increase in antiserum concentration resultsin an increase in antigen (HbAlc) precipitation, thus demonstrating thatthe antiserum of the invention reacts with the glycosylated N-terminalend of the HbAlc antigen.

Further, the antiserum can be demonstrated to be substantially free ofcross-reactivity to forms of hemoglobin other than HbAlc, as by thewell-known RIA dilution technique, using "cold" (unlabeled) HbAlc, HbAo,etc.

Immunoassay for HbAlc

The highly specific antibodies of the invention, obtained as describedabove, may be used for quantitatively detecting the presence of HbAlc ina blood sample by any of various suitable immunoassay techniques,including RIA, EIA and fluorescent immunoassay.

In such an assay, substantially pure HbAlc comprises the index antigen,which can be labeled radioactively (e.g. as with iodine-125, as shown inExample 6, above), or with an enzyme capable of quantitative detection,or with a fluorescein label.

In a preferred embodiment of RIA, a sample is reacted with an antibodymade according to the invention which is highly specific against HbAlcbut which is substantially free of cross-reactivity against the humanhemoglobin Ao, Ala and Alb, to produce an antibody-antigen (i.e.antibody-HbAlc) complex. The presence of the complex is thenquantitatively determined by radioimmunoassay by reacting the complexwith radioactive labeled HbAlc, or with labeled antigen of theinvention.

The reactions between the labeled HbAlc and the antibody-HbAlc complexcan conveniently be carried out at room temperature to about 37° C., forabout one-half hour.

The foregoing immunoassay methods, specifically including the RIA methoddetailed above, are useful in assessing carbohydrate intolerance in aliving mammal. In such an assessment, the presence of HbAlc in a sampleof the mammal's blood is quantitatively determined. Levels in excess ofabout 6% are indicative of carbohydrate intolerance.

Diagnostic kits for quantitatively assessing HbAlc levels in a samplemay comprise a container of the inventive antibody, in combination witha labeled (e.g. radioactively labeled) amount of substantially pureHbAlc for use as an antigen control. Labeling is preferably withiodine-125. The antibody may be packaged in either solution form, orlyophilized form suitable for reconstitution. Labeled antigen of theinvention can alternatively be used as the control.

The foregoing detailed description is given for clearness ofunderstanding only, and no unnecessary limitations are to be inferredtherefrom, as modifications will be obvious to those skilled in the art.

We claim:
 1. A method of preparing an antigen useful in obtainingantibodies against HbAlc, which antibodies are substantially free ofcross-reactivity to nonglycosylated hemoglobin, said method comprisingthe steps of:(a) synthesizing a hapten consisting of a peptide havingthe formula Val--His--Leu--Y wherein Y is chosen from the groupconsisting of Thr--, Thr--Pro--, Thr--Pro--Glu--, Thr--Pro--Glu--Glu--,Thr--Pro--Glu--Glu--Lys--, Thr--Pro--Glu--Glu--Lys--Ser--, andThr--Pro--Glu--Glu--Lys--Ser--Ala--, said hapten thereby having an aminoacid sequence corresponding to the NH₂ terminus of the hemoglobinβ-chain; (b) reacting the peptide of step (a) with glucose to obtain aglycosylated peptide having the formula

    glucose--Val--His--Leu--Y;

and (c) conjugating the glycosylated peptide of step (b) with a proteinto form an antigen suitable for injection into a host animal whosemetabolism does not naturally produce HbAlC for production of antibodiesthereto.
 2. The method of claim 1 wherein said protein is animmunoglobulin.
 3. The method of claim 2 wherein said protein comprisesIgG.
 4. The method of claim 1 wherein said peptide is covalently linkedto said protein.
 5. The method of claim 4 wherein said peptide isconjugated to said protein through a carbodiimide linkage.
 6. The methodof claim 2 wherein said peptide comprisesd-glucose--Val--His--Leu--Thr--Pro--Glu--Glu--, said protein is bovineIgG, and said peptide is covalently linked to said bovine IgG through acarbodiimide linkage.